抗氧化乐果棉蚜谷胱甘肽S-转移酶研究
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摘要
本研究以棉蚜(Aphis gossypii Glover)为实验材料，针对其广泛发生的抗药性问题，通过持续筛选建立棉蚜实验室种群，以昆虫体内重要的抗药性相关代谢酶系为切入点，从生理生化和分子生物学方面入手，明确了棉蚜体内与氧化乐果抗药性相关的酶系的变化以及抗性品系棉蚜体内谷胱甘肽S-转移酶（GSTs）的特征。
通过单头汰选得到了棉蚜氧化乐果敏感品系，其对氧化乐果的敏感性达到LC50值为18.23mg/L。采用浸叶法持续筛选建立了棉蚜氧化乐果抗性品系，其LC50值高达5657.90mg/L，抗性倍数为310.76。
通过对棉蚜体内重要的代谢酶系羧酸酯酶（CarE）、乙酰胆碱酯酶(AChE)的生化测定，没有发现CarE在棉蚜氧化乐果抗性形成中的显著变化。与敏感品系相比，抗性品系AChE对氧化乐果的敏感性出现了4.12倍的降低，其代谢碘化硫代乙酰胆碱的比活力由2.910.08mOD/mg pr. min下降到0.370.02mOD/mg pr. min，相应地，Vmax由11.030.21nmol/mg pr. min下降到了0.42
0.00nmol/mg pr. min，Km则由1.970.11μM降低到了0.610.01μM。说明乙酰胆碱酯酶的敏感性降低是棉蚜氧化乐果抗性形成的重要机制之一。
以1-氯2,4–二硝基苯为底物对GST_s进行生化测定，抗性品系GST_s比活力轻微下降，由176.54±7.21nmol/mg pr. min降至144.07±5.19nmol/mg pr. min，但Vm由250.00±9.90nmol/mg pr. min增至833.33±33.62nmol/mg pr. min，Km也由1.38±0.05mM升高到3.33±0.12mM。通过马来酸二乙酯（DEM）的体内抑制测定发现DEM对抗性品系棉蚜的增效比为0.11，与氧化乐果有拮抗作用，而体外抑制结果表明DEM对抗性品系GST_s的抑制能力有微弱降低，抑制中浓度（IC50）是敏感品系的1.17倍。对底物的代谢机制方面，敏感品系GST_s表现为底物浓度依赖的有序机制和乒乓机制相结合的复合机制，而抗性品系GST_s则表现为单一高效的有序机制。克隆得到了棉蚜Delta类GST_s的一个包含完整编码区的基因片段，推导其编码一个具有216个氨基酸残基的蛋白，其序列在敏感和抗性品系中没有差异。对该基因进行Real-time PCR验证，在抗性品系中，该基因的DNA拷贝数比敏感品系减少了1.52倍，而mRNA转录水平更是下调至敏感品系的0.33倍。以上结果表明在长期的氧化乐果选择压力下抗性品系GST_s在基因表达和代谢反应方面都发生了一定的适应性改变。
以上研究结果表明：抗性棉蚜GST_s在氧化乐果选择压力下，总活力受基因水平的调控而下降，但是其代谢机制由灵活性更强的复合机制转变为了催化效率更高的单一机制，单位GST_s的代谢能力升高，保证了抗性品系棉蚜体内GST_s正常的生理功能。研究结果为棉蚜的氧化乐果抗性机制研究提供了有力的理论依据。
Cotton aphid, Aphis gossypii Glover (Homoptera： Aphididae), emergesresistance nearly to all of the commercialized insecticide classes globally. In order toelucidate the resistant mechanism to omethoate in cotton aphid, two aphid strainshave been built in laboratory. The researches focus on the important enzymes whichare highly related to insecticide resistance in cotton aphid. Biochemical andmolecular methods were conducted, and the results revealed the adaptation toomethoate stress of cotton aphid.
The susceptible strain exhibited a low LC50of omethoate as18.23mg/L. Andthe resistant strain had developed a high level resistance to omethoate by frequentlyselection. The LC50of omethoate as high as5657.90mg/L, and the resistant foldwas310.76when compared to the susceptible strain.
The biochemical results of carboxylesterase（CarE）indicated that it was notinvolved in the omethoate resistance of cotton aphid. Compared with the susceptiblestrain, the susceptibility to omethoate of acetylcholinesterase (AChE) downregulatedof4.12-fold in the resistant strain. The special activity to acetylthiocholine iodidereduced from2.91±0.08to0.370.02mOD/mg pr. min. Correlatively, the Vmaxreduced from11.030.21to0.420.00nmol/mg pr. min, and the Kmreduced from1.970.11to0.610.01μM. So the downregulation of AChE susceptibility wasone of the important resistant mechanisms to omethoate in cotton aphid.
1-ch1oro-2,4-dinitrobenzene (CDNB) was applied to determine the activity ofglutathione S-transferases (GST_s). The special activity was144.07±5.19nmol/mgpr. min in the resistant strain that slightly lower when compared to the susceptiblestrain which possessed higher activity of176.54±7.21nmol/mg pr. min. But theVmaxelevated from250.00±9.90to833.33±33.62nmol/mg pr. min and Kmelevatedfrom1.38±0.05to3.33±0.12mM. Thediethyl malate (DEM) was an antagonism with omethoate as the synergism ratio was0.11. The IC50to the resistant strain(2.32×10-4±0.14μM) was slightly higher (1.17-fold) than the susceptible strain(1.98×10-4±0.13μM). GST_s from the two strains exhibited differential catalyticmodels. In the susceptible strain, a biphasic kinetic mechanism was observed with abreakpoint depend on the GSH concentration, in the higher GSH concentrationcondition, GST_scatalyzed GSH conjugated with CDNB by a sequential mechanismand a ping-pong pathway predominated the conjugation in the lower GSHconcentration range. However, in the resistant strain, the single efficiently sequentialmechanism constantly catalyzed GSH conjugated with CDNB. A Delta GST_sDNAfragment was successfully amplified from the susceptible and resistant strainrespectively, which encoded the identical full GST_sprotein sequence (216aminoacid residues). The GST_sDNA copies was1.52-fold lower in the resistant strainwhile the mRNA transcription level was much lower (0.33-fold). It was assumedthat the GST_smay become insensitivity and was been compensated by efficientsingle ordered sequential mechanism in the high level omethoate resistance of cottonaphid.
The researches provided evidences for the effect of AChE in omethoateresistance of cotton aphid, and detected the modification of GST_s under theomthoate stress. Which were important to elucidate the mechanism of omethoateresistance in A. gossypii.
通过单头汰选得到了棉蚜氧化乐果敏感品系，其对氧化乐果的敏感性达到LC50值为18.23mg/L。采用浸叶法持续筛选建立了棉蚜氧化乐果抗性品系，其LC50值高达5657.90mg/L，抗性倍数为310.76。
通过对棉蚜体内重要的代谢酶系羧酸酯酶（CarE）、乙酰胆碱酯酶(AChE)的生化测定，没有发现CarE在棉蚜氧化乐果抗性形成中的显著变化。与敏感品系相比，抗性品系AChE对氧化乐果的敏感性出现了4.12倍的降低，其代谢碘化硫代乙酰胆碱的比活力由2.910.08mOD/mg pr. min下降到0.370.02mOD/mg pr. min，相应地，Vmax由11.030.21nmol/mg pr. min下降到了0.42
0.00nmol/mg pr. min，Km则由1.970.11μM降低到了0.610.01μM。说明乙酰胆碱酯酶的敏感性降低是棉蚜氧化乐果抗性形成的重要机制之一。
以1-氯2,4–二硝基苯为底物对GST_s进行生化测定，抗性品系GST_s比活力轻微下降，由176.54±7.21nmol/mg pr. min降至144.07±5.19nmol/mg pr. min，但Vm由250.00±9.90nmol/mg pr. min增至833.33±33.62nmol/mg pr. min，Km也由1.38±0.05mM升高到3.33±0.12mM。通过马来酸二乙酯（DEM）的体内抑制测定发现DEM对抗性品系棉蚜的增效比为0.11，与氧化乐果有拮抗作用，而体外抑制结果表明DEM对抗性品系GST_s的抑制能力有微弱降低，抑制中浓度（IC50）是敏感品系的1.17倍。对底物的代谢机制方面，敏感品系GST_s表现为底物浓度依赖的有序机制和乒乓机制相结合的复合机制，而抗性品系GST_s则表现为单一高效的有序机制。克隆得到了棉蚜Delta类GST_s的一个包含完整编码区的基因片段，推导其编码一个具有216个氨基酸残基的蛋白，其序列在敏感和抗性品系中没有差异。对该基因进行Real-time PCR验证，在抗性品系中，该基因的DNA拷贝数比敏感品系减少了1.52倍，而mRNA转录水平更是下调至敏感品系的0.33倍。以上结果表明在长期的氧化乐果选择压力下抗性品系GST_s在基因表达和代谢反应方面都发生了一定的适应性改变。
以上研究结果表明：抗性棉蚜GST_s在氧化乐果选择压力下，总活力受基因水平的调控而下降，但是其代谢机制由灵活性更强的复合机制转变为了催化效率更高的单一机制，单位GST_s的代谢能力升高，保证了抗性品系棉蚜体内GST_s正常的生理功能。研究结果为棉蚜的氧化乐果抗性机制研究提供了有力的理论依据。
Cotton aphid, Aphis gossypii Glover (Homoptera： Aphididae), emergesresistance nearly to all of the commercialized insecticide classes globally. In order toelucidate the resistant mechanism to omethoate in cotton aphid, two aphid strainshave been built in laboratory. The researches focus on the important enzymes whichare highly related to insecticide resistance in cotton aphid. Biochemical andmolecular methods were conducted, and the results revealed the adaptation toomethoate stress of cotton aphid.
The susceptible strain exhibited a low LC50of omethoate as18.23mg/L. Andthe resistant strain had developed a high level resistance to omethoate by frequentlyselection. The LC50of omethoate as high as5657.90mg/L, and the resistant foldwas310.76when compared to the susceptible strain.
The biochemical results of carboxylesterase（CarE）indicated that it was notinvolved in the omethoate resistance of cotton aphid. Compared with the susceptiblestrain, the susceptibility to omethoate of acetylcholinesterase (AChE) downregulatedof4.12-fold in the resistant strain. The special activity to acetylthiocholine iodidereduced from2.91±0.08to0.370.02mOD/mg pr. min. Correlatively, the Vmaxreduced from11.030.21to0.420.00nmol/mg pr. min, and the Kmreduced from1.970.11to0.610.01μM. So the downregulation of AChE susceptibility wasone of the important resistant mechanisms to omethoate in cotton aphid.
1-ch1oro-2,4-dinitrobenzene (CDNB) was applied to determine the activity ofglutathione S-transferases (GST_s). The special activity was144.07±5.19nmol/mgpr. min in the resistant strain that slightly lower when compared to the susceptiblestrain which possessed higher activity of176.54±7.21nmol/mg pr. min. But theVmaxelevated from250.00±9.90to833.33±33.62nmol/mg pr. min and Kmelevatedfrom1.38±0.05to3.33±0.12mM. Thediethyl malate (DEM) was an antagonism with omethoate as the synergism ratio was0.11. The IC50to the resistant strain(2.32×10-4±0.14μM) was slightly higher (1.17-fold) than the susceptible strain(1.98×10-4±0.13μM). GST_s from the two strains exhibited differential catalyticmodels. In the susceptible strain, a biphasic kinetic mechanism was observed with abreakpoint depend on the GSH concentration, in the higher GSH concentrationcondition, GST_scatalyzed GSH conjugated with CDNB by a sequential mechanismand a ping-pong pathway predominated the conjugation in the lower GSHconcentration range. However, in the resistant strain, the single efficiently sequentialmechanism constantly catalyzed GSH conjugated with CDNB. A Delta GST_sDNAfragment was successfully amplified from the susceptible and resistant strainrespectively, which encoded the identical full GST_sprotein sequence (216aminoacid residues). The GST_sDNA copies was1.52-fold lower in the resistant strainwhile the mRNA transcription level was much lower (0.33-fold). It was assumedthat the GST_smay become insensitivity and was been compensated by efficientsingle ordered sequential mechanism in the high level omethoate resistance of cottonaphid.
The researches provided evidences for the effect of AChE in omethoateresistance of cotton aphid, and detected the modification of GST_s under theomthoate stress. Which were important to elucidate the mechanism of omethoateresistance in A. gossypii.
引文
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